title: Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies.
abstract: Here, we report a method, adapted to the human epidermis, allowing isolation of desmosomes in small tissue fractions. The methods previously developed for animal skin did not work efficiently with human tissue. Enrichment of desmosomes was performed by the association of two incubation steps in acidic solutions containing detergent NP-40 at two different concentrations followed by a sonication step. The suspension was centrifuged twice: first to remove the heavy cell fragments and then at 16000 g on a discontinuous sucrose gradient. A desmosome-enriched fraction (DsF) was collected at the 30-50% sucrose interface. We demonstrate by immunoelectron microscopy and by western blotting that the central part of the desmosome structure is preserved as well as the antigenicity of its components. Our approach, allowing a significant enrichment of the cell fractions containing desmosomes, can be used to immunize animals and create new antibodies directed against desmosomal components. Using this strategy, new and so far poorly studied molecules incorporated into the desmosome cores could be targeted more easily.
authors.affiliation: EA4169/Normal and pathological functions of skin barrier, Edouard Herriot Hospital, University Lyon 1, Lyon, France. yongoua.sandjeu@gmail.com
authors.forename: Yongoua
authors.initials: Y
authors.lastname: Sandjeu
authors.forename: Sylvie
authors.initials: S
authors.lastname: Callejon
authors.forename: Claude
authors.initials: C
authors.lastname: Vincent
authors.forename: Marek
authors.initials: M
authors.lastname: Haftek
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D003896:Desmosomes; D000078404:Epidermal Cells; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron